Plasmids are genetic elements essentially found in bacteria, made up of a molecule of deoxyribonucleic acid, which is most commonly circular and the replication of which is autonomous and independent from that of genomic DNA. Natural plasmids isolated from a very broad variety of bacteria are capable of accomplishing several cellular functions. The first, which is vital for all plasmids, is that responsible for their replication, generally carried out in a manner which is synchronized with replication of the genomic DNA and cell division. Besides the region required for replication of the plasmid, all natural plasmids carry genes which encode proteins, the function of which most commonly remains unknown due to a lack of scientific investigation with regard to these genes. The number of genes present on a plasmid determines the size of this plasmid, the smallest natural plasmids containing only two to three genes. The properties of plasmids attracted research scientists to them very early on, to make them vehicles for transporting and expressing genes in prokaryotic cells, as well as in eukaryotic cells. The very rapid progress observed in the fields of the molecular biology of nucleic acids and proteins, over the past two decades, can in part be attributed to the exploitation of recombined plasmids constructed from fragments of natural DNA of plasmid origin or other cellular DNAs, and even chemically synthesized.
The four bases adenine (A), guanine (G), cytosine (C) and thymine (T) which constitute deoxyribonucleic acid (DNA) are distributed in 16 dinucleotide configurations, namely CG, GC, TA, AT, CC, GG, TT, AA, TG, CA, AG, CT, AC, GT, GA and TC. Analysis of the qualitative distribution of the dinucleotides of the DNA of thousands of plasmids for which sequences are known reveals that the 16 dinucleotides are always present in all natural plasmids or plasmids constructed in the laboratory. However, analysis of the quantitative distribution of the dinucleotides of plasmids shows great disparities which depend, only partly, on the percentage of each one of the four bases of the DNA. Specifically, comparison of the frequencies observed for each one of the dinucleotides with those of the frequencies calculated on the basis of a random association between two bases, for a given plasmid, can demonstrate major differences for several dinucelotides in terms of an over representation, or, on the contrary, an under representation (Campbell A., Mrazek J. and Karlin S. (1999) Proc Natl Acad Sci USA 96, 9184-9). The differences observed in the distribution of certain dinucleotides, not always the same, of natural plasmids isolated from bacteria of phylogenically distant species have been explained by differences in specificity in the mechanisms of repair, recombination and replication acting on the cellular DNAs.
Gene transfers in vitro into cells in culture and in vivo into various animals are common practices undergoing great development, on the one hand, for the purpose of gaining a better understanding of cell function and, on the other hand, in order to apply these techniques to cell and gene therapies. None of the viral vectors and plasmid vectors among the panoply of vectors available for gene transfer in animals has taken a decisive advantage over the others, since each one has advantages, but also disadvantages. There is, however, an application in which naked plasmid DNA or plasmid DNA complexed with various substances to facilitate DNA transport to the nucleus is the subject of intense research activity, namely that of immunizing DNA. The principle of immunizing DNA is based on the immune responses observed in laboratory animals treated, by intramuscular or intradermal injection or by inhalation, with plasmid DNA encoding an antigenic peptide. It is now well established that a first consequence of introducing a plasmid DNA derived from the bacteria E. coli into the body of an experimental animal intravenously and intramuscularly is the rapid production of various cytokines by the guard cells of the immune system (Krieg A. M. and Kline J. N. (2000) Immunopharmacology 48, 303-305). This response is extremely specific for bacterial DNA since DNA extracted from animal cells does not cause such an induction of cytokines under the same conditions. The cellular mechanisms involved in this immune response are far from being fully understood. However, it is known that the recognition which discriminates between bacterial DNA and DNA of animal origin takes place at the level of structural differences relating to the methylation of certain cytosines of the molecule. Specifically, mammalian DNA is naturally methylated at the cytosine of all CG dinucleotides (subsequently written CpG), with the exception of short regions of high CpG density, called CpG islands, present in functional regions in some promoters. DNA extracted from E. coli does not exhibit this type of methylation due to the absence of the enzyme activity capable of accomplishing this modification in this bacterium. It is, however, possible to methylate the CpGs of plasmid DNA extracted from E. coli in a test tube with an appropriate enzyme. Under these conditions, DNA methylated in vitro loses a great deal of its immunostimulant activity compared to control nonmethylated DNA. The E. coli strain K12, from which virtually all the mutant strains used to produce plasmid DNAs are derived, contains an enzyme activity (DNA methylase dcm (Palmer B. R. and Marinus M. G. (1994) Gene 143, 1-12)) which leads to methylation of cytosine occurring in the nucleic acid context CC(A/T)GG. All plasmid vectors for gene transfer contain this sequence in varying number and, as a result, their DNA molecule contains methylated cytosines which are not found in mammalian DNA. This form of methylation specific to E. coli thus introduces another difference into the cytosine methylations between bacterial DNA and that of mammals, which might contribute to the immunostimulant capacity of plasmid DNA.
The CpG frequency in primate and rodent DNAs is, overall, much lower than that expected on the basis of the frequency of cytosines and guanines. The CpG deficiency is dependent, for a given DNA fragment, on the biological role of this fragment, intergenic regions containing only a fifth of the expected frequency, while exons have a less marked deficiency and, at the other extreme, some promoters containing a large CpG island exhibit a CpG percentage close to that expected. Analysis of the data from sequencing human cDNAs and chromosomes reveals, however, broad heterogeneities in the CpG frequency for promoter regions and cDNAs. This observation is illustrated by the cDNA of the human gene encoding interleukin 2, which has just one CpG. Similarly, a portion of the promoter of this gene containing the TATA box does not contain any CpG, but, on the other hand, the upstream portion rich in transcription factor recognition sites contains CpGs. The regions positioned 3′ of the genes, formed by the 3′ UTRs (untranslated regions) and the polyadenylation and end of transcription sequences are rather poor in CpG. In humans, it is not unusual to find regions immediately downstream of the genes, which are devoid of CpG. However, the human sequencing data available at the end of 2000 have not made it possible to demonstrate a single transcriptional unit, made up of the transcription promoter regions, a gene with or without an intron and the polyadenylation region, which is completely devoid of CpG. The situation of the CpGs in E. coli is quite different from that of animal cells since the frequency of CpGs in the genomic DNA of this bacterium is slightly greater compared to the calculated frequency. The same is true for the CpGs of natural plasmids isolated from hospital strains of E. coli. The recombined plasmids resulting from genetic manipulations, used for gene transfer, exhibit variations in their CpG numbers which depend on the origin of the fragments inserted into the vector. Analysis of the sequences of several tens of recombined E. coli plasmids randomly taken from the GenBank databank shows that the plasmids most lacking in CpG have, at the very most, a 50% deficiency in the number of their CpGs.
As regards the present invention, it provides products and methods for synthesizing plasmid DNA in E. coli which is completely devoid of CpG and in which the cytosines placed in the context CC(A/T)GG are not methylated. To the applicant's knowledge, this is the first description of such products which exhibit such a structure while at the same time having conserved their function.